Regulation of Photosynthetic GAPDH Dissectedby Mutants1

Francesca Sparla2, Mirko Zaffagnini2, Norbert Wedel3, Renate Scheibe, Paolo Pupillo, and Paolo Trost*

Laboratory of Molecular Plant Physiology, Department of Biology, University of Bologna, 40126 Bologna,Italy (F.S., M.Z., P.P., P.T.); and Department of Plant Physiology, Faculty of Biology and Chemistry,University of Osnabrueck, D–49069 Osnabrueck, Germany (N.W., R.S.)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants catalyzes an NADPH-consuming reaction, which ispart of the Calvin cycle. This reaction is regulated by light via thioredoxins and metabolites, while a minor NADH-dependentactivity is constant and constitutive. The major native isozyme is formed by A- and B-subunits in stoichiometric ratio (A2B2,A8B8), but tetramers of recombinant B-subunits (GapB) display similar regulatory features to A2B2-GAPDH. The C-terminalextension (CTE) of B-subunits is essential for thioredoxin-mediated regulation and NAD-induced aggregation to partiallyinactive oligomers (A8B8, B8). Deletion mutant B(minCTE) is redox insensitive and invariably tetrameric, and chimeric mutantA(plusCTE) acquired redox sensitivity and capacity to aggregate to very large oligomers in presence of NAD. Redox regulationprincipally affects the turnover number, without significantly changing the affinity for either 1,3-bisphosphoglycerate orNADPH. Mutant R77A of GapB, B(R77A), is down-regulated and mimics the behavior of oxidized GapB under any redoxcondition, whereas mutant B(E362Q) is constantly up-regulated, resembling reduced GapB. Despite their redox insensitivity,both B(R77A) and B(E362Q) mutants are notably prone to aggregate in presence of NAD. Based on structural data and currentfunctional analysis, a model of GAPDH redox regulation is presented. Formation of a disulfide in the CTE induces a con-formational change of the GAPDH with repositioning of the terminal amino acid Glu-362 in the proximity of Arg-77. The latterresidue is thus distracted from binding the 2#-phosphate of NADP, with the final effect that the enzyme relaxes to a con-formation leading to a slower NADPH-dependent catalytic activity.

Since the discovery of the role of the ferredoxin/thioredoxin system in regulating photosynthetic car-bon assimilation (for review, see Buchanan et al., 2002),the kinetic regulation of enzyme activity by redoxsignaling has attracted the interest of plant physiolo-gists. Recently, the number of known thioredoxintargets in plants has been increasing thanks to newmethods for the identification of thioredoxin-interactingproteins (Motohashi et al., 2001; Yano et al., 2001;Marchand et al., 2004). As a result, many metabolicpathways besides the Calvin cycle are now believed tobe modulated by thioredoxins in plants (Buchananand Balmer, 2005). These small ubiquitous proteinscontain a conserved active site with the sequenceWC[G/P]PC by means of which they control the redoxstate of target enzymes via dithiol-disulfide exchangereactions and regulate enzyme activities in a sensitiveand reversible way in relation to the redox state of thecell. Compared to bacteria and animals, plants containa large variety of thioredoxins localized in chloro-plasts, mitochondria, and cytoplasm (Johnson et al.,1987; Laloi et al., 2001); e.g. nine plastidial thioredoxin

forms with distinct target specificities have been char-acterized in Arabidopsis (Arabidopsis thaliana; Collinet al., 2003, 2004).

At difference from thioredoxins, thioredoxin targetshave no consensus sequences, and it is still unclearwhether the molecular mechanisms of thioredoxinregulation follow any general rule. Based on crystalstructures of reduced/oxidized forms and the kineticanalysis of site-specific mutants, the redox regulationsof Fru bisphosphate phosphatase and NADP-malatedehydrogenase were satisfactorily explained at themolecular level and found to be different (Carr et al.,1999; Chiadmi et al., 1999; Johansson et al., 1999). Noother thioredoxin-regulated enzyme has been investi-gated in such depth.

Photosynthetic glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) is a target of thioredoxins inplant chloroplasts (Wolosiuk and Buchanan, 1978;Sparla et al., 2002). Two isoforms of chloroplastGAPDH exist in higher plants, an abundant hetero-meric AB-isoform and a minor homotetrameric A4-isoform, with quite different regulatory properties(Cerff, 1979; Brinkmann et al., 1989; Ferri et al., 1990;Scagliarini et al., 1993). A4-GAPDH is regulatedthrough the interaction with the small protein CP12,giving a supramolecular complex with phosphoribu-lokinase (Wedel and Soll, 1998; Graciet et al., 2003).Formation and dissociation of this supramolecularcomplex contributes to light-dependent modulationof both enzyme activities and hence to the overallregulation of photosynthetic metabolism (Scheibe

1 This work was supported by Ministero dell’Istruzione,dell’Universita e della Ricerca (PRIN 2003).

2 These authors contributed equally to the paper.3 Present address: Julius-Leber-Str. 27, 24145 Kiel, Germany.* Corresponding author; e-mail trost@alma.unibo.it; fax 39–051–

242576.Article, publication date, and citation information can be found at

www.plantphysiol.org/cgi/doi/10.1104/pp.105.062117.

2210 Plant Physiology, August 2005, Vol. 138, pp. 2210–2219, www.plantphysiol.org � 2005 American Society of Plant Biologists

et al., 2002; Graciet et al., 2004). The crystal structureof spinach (Spinacia oleracea) A4-GAPDH in complexwith either NADP or NAD has recently been solved(Fermani et al., 2001; Falini et al., 2003). It was arguedthat Arg-77 and Ser-188, by specifically interacting withthe 2#-phosphate of NADP, are key residues for co-enzyme specificity in this protein (Sparla et al., 2004).

Although AB-GAPDH may also participate in su-pramolecular complexes with CP12 and phospho-ribulokinase (Clasper et al., 1991; Wedel et al., 1997;Scheibe et al., 2002), AB-GAPDH exhibits in additionan autonomous type of regulation (Pupillo andGiuliani Piccari, 1975), which depends on the presenceof a C-terminal extension (CTE) characteristic ofB-subunits and homologous to the C-terminal end ofCP12 (Baalmann et al., 1996; Pohlmeyer et al., 1996).The enzyme activity is modulated through the re-versible formation of a disulfide bridge in the CTEbetween Cys-349 and Cys-358, operated by thiore-doxin f in relation to the light/dark cycle (Sparla et al.,2002). Redox regulation of AB-GAPDH selectively af-fects the turnover number of the NADPH-dependentactivity, leaving unchanged the alternative activitywith NADH as coenzyme. A comparable decrease ofNADPH-dependent activity was recently obtained inrecombinant A4-GAPDH impaired in coenzyme rec-ognition due to the substitution of Ser-188 into Ala.The data thus suggest a link between coenzyme speci-ficity and redox regulation of the GAPDH (Sparla et al.,2004).

With the aim of unraveling the molecular mecha-nism of thioredoxin regulation in AB-GAPDH, herewe report on a series of mutants affected in redoxregulation. By integrating information derived fromkinetic analysis of these mutants and tridimensionalstructures of A4-GAPDH, a molecular model of AB-GAPDH regulation by thioredoxin was derived. Giventhe similarity between CP12 and CTE, this modelprovides a molecular basis for a broader understand-ing of photosynthetic GAPDH regulation includingthe CP12-dependent regulation of A4-type isozymes.

RESULTS

Protein Engineering, Heterologous Expression,and Purification of Regulatory Mutants of

Photosynthetic GAPDH

Of the two subunits present in the AB-type photo-synthetic GAPDH, subunit-B contains the autoinhibi-tory domain CTE that confers regulatory properties tothe whole enzyme. Different site-specific mutants ofGAPDH B-subunits, i.e. B(S188A), B(R77A), andB(E362Q), were constructed and expressed in Escher-ichia coli (Fig. 1). The rationale behind the choice ofmutations S188A and R77A in GapB was that bothresidues are involved in NADP versus NAD recogni-tion in recombinant A4-GAPDH (Sparla et al., 2004)and, therefore, are possible candidates for NADPH-

specific regulation of GapB. Glu-362, the C-terminalamino acid of B-subunits, was mutated into Gln withthe aim of decreasing the overall negative charge of theCTE domain. This domain was suggested to makeelectrostatic interactions with the positively chargedS-loop (Asp-181 to Ala-199), which binds NADP viaSer-188 (Fig. 1; Reichert et al., 2000; Sparla et al., 2002).The chimeric mutant A(plusCTE) and deletion mutantB(minCTE) (Baalmann et al., 1996) were also construc-ted and expressed in E. coli as a further test for theregulatory function of CTE.

All recombinant proteins, including wild-typeGapA and GapB isoforms, were purified to homoge-neity by two consecutive ion-exchange chromato-graphic steps. Elution gradients were optimized fordifferent GAPDH forms. Yields were in the range of1 mg of pure protein per liter of liquid culture for anyform except B(R77A) whose recovery after purificationwas less than 50 mg per liter. Native AB-GAPDH wasalso purified from spinach leaves and tested forcomparison. After purification, the maximal, fullyactivated NADPH-dependent specific activity of anyGAPDH form here described was in the range of 80 to130 mmol min21 mg21, with the notable exception ofmutant B(R77A) whose maximum specific activity was46 mmol min21 mg21.

Sensitivity of Regulatory Mutants to the RedoxState of Thioredoxin

The regulation of spinach AB-GAPDH activity bythioredoxin f was previously shown to depend onB-subunits and to specifically affect the NADPH-dependent activity (Em,7.9 2353 6 11 mV; Fig. 2;Baalmann et al., 1996; Li and Anderson, 1997; Sparlaet al., 2002). Redox modulation was not due to a changein affinity for substrate (1,3-bisphosphoglycerate) orcoenzyme (NADPH), but rather to a 2.2-fold shift inmaximal catalytic activity (Table I; Sparla et al., 2004).As a consequence, redox titrations of enzyme activitiesmeasured with standard assay (Fig. 2) approximatelyindicate specific changes in Vmax.

In agreement with the regulatory role of B-subunits,homotetrameric A4-GAPDH was redox-insensitive. Incontrast, the redox sensitivity of recombinant GapB(Em,7.923576 12 mV) was similar to native AB-GAPDH(Fig. 2). The affinity of either recombinant GapA orGapB for NADPH [Km(NADPH) about 30 mM] was slightlyhigher than in native AB-GAPDH [Km(NADPH) 50 mM],with negligible differences between GapB reduced/oxidized forms. Km(BPGA) averaged 20 mM in GapA,GapB, or AB-GAPDH under any redox conditions(Table I), supporting the conclusion that redox regula-tion of B-containing GAPDH forms essentially consistsin a Vmax effect, i.e. a 2- to 3-fold decrease of Vmax inoxidized versus reduced forms.

The involvement of CTE in the regulatory mecha-nism was confirmed by the redox insensitivity ofB(minCTE) deletion mutant (Fig. 2; Li and Anderson,1997; Sparla et al., 2002). On the other hand, the newly

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described chimeric mutant A(plusCTE) gained redoxsensitivity as expected (Fig. 2). However, despite theextensive sequence homology between GapA andGapB (CTE excluded), the behavior of mutantA(plusCTE) was different from GapB in that bothNADPH- and NADH-dependent activities werefound to be similarly redox-modulated (Fig. 2) anddecreased upon enzyme oxidation to about 50% of thefully reduced enzyme. Midpoint redox potentials atpH 7.9 were 2340 6 14 mV and 2345 6 18 mV for theNADPH- and the NADH-dependent activities, respec-

tively, slightly less negative than AB-GAPDH andrecombinant GapB. The affinity of A(plusCTE) forBPGA [Km(BPGA) about 20 mM] was as in GapA regard-less of the redox state (Table I), that for NADPH wassomewhat lower [Km(NADPH) 65 mM reduced, 43 mM

oxidized]. Albeit not identical to GapB, the redoxsensitivity of A(plusCTE) clearly shows the capabilityof recombinant CTE to modulate the activity of non-regulatory GAPDH isoforms.

The site-specific mutant B(S188A) was also charac-terized by redox sensitivity of both NADPH- and

Figure 1. Amino acid sequence and schematicrepresentation of wild-type and mutant formsstudied in this work. A, Pairwise alignment ofGapA and GapB subunits of spinach GAPDH.Residues are numbered according to the structureof Bacillus stearothermophilus GAPDH depos-ited in the Protein Data Bank (Biesecker et al.,1977). Insertion or deletions with respect to B.stearothermophilus GAPDH are underlined.Amino acids identical in both GapA and GapBare on a gray field. Site specific mutations of Arg-77, Ser-188, and Glu-362 are on a black field.Sequences corresponding to the S-loop and theCTE are double underlined, and charged aminoacids within these sequences are indicated bysymbols1 and2. The regulatory disulfide bridgebetween Cys-349 and Cys-358 (Sparla et al.,2002) is indicated. B, Schematic survey of allGAPDH forms studied in this work. GapA isrepresented by a gray rectangle, GapB by a whiterectangle (positions 1–334), plus a black rectan-gle corresponding to the CTE (positions 335–362). Site-specific substitutions are reported inbold.

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NADH-dependent activities (Fig. 2). Midpoint redoxpotentials for both reactions were less negative than allother redox-sensitive GAPDH forms (NADPH, Em,7.92329 6 7 mV; NADH, Em,7.9 2346 6 14 mV). Redoxmodulation of this mutant was associated with aslightly lower affinity for the substrates [Km(BPGA)

about 31 mM; Km(NADPH) about 46 mM, reducing con-ditions; Table I].

Crystal structures of recombinant GapA showedthat the extra 2#-phosphate group of NADP is stabi-lized by Arg-77 besides Ser-188 (Sparla et al., 2004).Consistent with this finding, substitution of an Ala forArg-77 in GapB resulted in a mutant protein withunique kinetic properties. While the activity of themutant with NADH was comparable to any otherGAPDH form (Vmax about 50 mmol min21 mg21), theNADPH activity was lowered and completely insensi-tive to the redox poise (Fig. 2) in consequence of twoindependent effects: (1) the affinity for NADPH mea-sured as Km was 2.5-fold lower than in wild-type GapB(Table I); and (2) Vmax(NADPH) under any redox condi-tion (46 mmol min21 mg21) was similar to Vmax(NADPH)of oxidized GapB (50 mmol min21 mg21) and in linewith NADH-dependent reaction rates. The R77A mu-tation introduced in GapB thus appears to block theprotein in a permanent unactivated state with catalyticproperties similar to oxidized GapB.

The autoinhibitory function of the CTE of GapB hasbeen proposed to be mediated by negative chargespresent in this mobile extension and an array ofpositive charges located on the S-loop (Fig. 1). Inmutant B(E362Q), the negatively charged side chain ofC-terminal Glu-362 was converted into a neutral sidechain (Gln). The mutation had no effect on the affinityfor substrate or coenzyme (Table I) but made B(E362Q)completely redox-insensitive. As a consequence, theNADPH activity remained at maximal levels underoxidizing conditions. In remarkable contrast toB(R77A), mutant B(E362Q) thus behaved as a constitu-tively ‘‘activated’’ form reproducing the kinetic prop-erties of reduced GapB (Fig. 2).

Figure 2. Redox titration of eight different wild-type and mutant formsof spinach GAPDH in the presence of spinach thioredoxin f. Activitieswere measured under standard conditions (see ‘‘Materials and Meth-ods’’) with either NADPH (black circles) or NADH (white circles) aselectron donor. All GAPDH forms displayed a maximal catalyticactivity with NADPH as coenzyme under reducing conditions in therange of 80 to 130 mmol min21 mg21, with the exception of mutantB(R77A) whose activity under these conditions was 46 mmol min21

mg21. In the graphs, activities under varying redox conditions havebeen normalized to the NADPH-activity of each form under fullyreducing conditions, which was set to 1 for all GAPDH forms withsimilar maximal activity (80–130 mmol min21 mg21) and set to 0.5 forB(R77A) (46 mmol min21 mg21).

Table I. Kinetic parameters of eight GAPDH forms obtained bysteady-state analysis of the NADPH-dependent reaction

Redox-sensitive forms [AB-GAPDH, GapB, B(S188A), andA(plusCTE)] were analyzed under both reducing and oxidizing con-ditions (3 h incubation with 20 mM reduced or oxidized dithiothreitoland spinach thioredoxin f ). Kinetic experiments were repeated three tosix times for each enzyme form. Data are presented as mean 6 SDs.

Enzyme FormOxidation

StateKm(NADPH) Km(BPGA)

VmaxðreducedÞVmaxðoxidizedÞ

mM mM

AB-GAPDHa Reduced 50 6 17 20 6 11 2.2 6 0.7Oxidized 50 6 12 24 6 1

GapB Reduced 30 6 4 15 6 2 2.2 6 0.7Oxidized 38 6 8 20 6 6

B(S188A) Reduced 46 6 14 31 6 7 3.1 6 0.7Oxidized 24 6 13 27 6 0

B(R77A) – 78 6 17 16 6 4 –B(E362Q) – 24 6 7 12 6 2 –B(minCTE) – 19 6 6 22 6 9 –GapAa – 29 6 8 15 6 5 –A(plusCTE) Reduced 65 6 21 21 6 10 2.1 6 0.6

Oxidized 43 6 2 19 6 14

aData from Sparla et al. (2004).

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Redox insensitivity of B(E362Q) was not due to theabsence of the regulatory disulfide in the oxidizedprotein. Oxidized B(E362Q) was in fact less reactivewith the thiol-labeling reagent monobromobimane bya factor of 1.7 6 0.1 in respect to the reduced protein,indicating that the mutation did not hamper theformation of the regulatory disulfide.

Quaternary Structures

It is well known that the aggregation state of nativeAB-GAPDH depends on the type of pyridine nucleo-tide bound to the protein (Pupillo and Giuliani Piccari,1975). In the presence of NADP, the tetrameric confor-mation A2B2 is stabilized (apparent molecular mass 189kD in size exclusion chromatography; Fig. 3), while inthe presence of NAD, the enzyme aggregates to anoligomer with 4-fold higher molecular mass (760 kD),stronglysuggestingA8B8 stoichiometry.TheCTEofGapBis required for NAD-mediated aggregation (Baalmannet al., 1996; Li and Anderson, 1997) and, in fact,B(minCTE) (151 kD) as well as GapA (132 kD) failedto aggregate in the presence of NAD and eluted as tetra-mers in the presence of either NAD or NADP (Fig. 3).

The chimeric protein A(plusCTE) also occurred asa tetramer in the presence of NADP, and its apparentsize (270 kD) was similar to wild-type GapB under thesame conditions (243 kD). Notably, all CTE-containingconstructs were found to be appreciably larger thanA2B2-GAPDH in size exclusion chromatography. Theactual molecular mass of tetrameric GapB is 157 kD,and the strong discrepancy between calculated andexperimental values may be ascribed to bulkiness ofthe CTE that is predicted to be exposed to the medium(Sparla et al., 2002). All GapB mutants, B(E362Q): 232

kD, B(S188A): 247 kD, B(R77A): 278 kD, in the pres-ence of NADP were basically similar in size to wild-type GapB, with an apparent correlation of Stokes’radius with increasing severity of the mutation.

In the presence of NAD, mutant A(plusCTE) gener-ated an unexpectedly high molecular mass form (.1.8MD), at least 7-fold bigger than the tetramer; Fig. 3).Under the same conditions, all GapB forms (wild typeand mutants) associated to oligomers ranging from491 (wild type) to 553 kD [B(S188A)], compatible withoctameric structures.

DISCUSSION

NADP-dependent photosynthetic GAPDHs arepresent in every organism featuring oxygenic photo-synthesis. Such archaic photosynthetic groups as cya-nobacteria, red algae, and green microalgae containa simple tetrameric NADP-GAPDH only constitutedby A-type subunits (Figge et al., 1999). In some of theseorganisms including Synechocystis PCC6803 (Wedeland Soll, 1998), Chlamydomonas reinhardtii (Gracietet al., 2003), and Scenedesmus obliquus (Nicholson et al.,1987), the regulation of GAPDH appears to involve thereversible formation of a supramolecular complexbetween GAPDH and phosphoribulokinase (PRK),mediated by the small photosynthetic protein CP12.

In higher plants and certain green algae (e.g. Co-leochaete and Chara), a second subunit of photosyn-thetic GAPDH (GapB) came on the scene as a likelyproduct of gene fusion between subunit-A and theC-terminal end of CP12 (Pohlmeyer et al., 1996). A pairof Cys in the CTE of GapB reversibly form an intra-chain disulfide under the control of thioredoxins.Disulfide-bearing CTE acts as an autoinhibitory do-main that gives rise to a further, autonomous mecha-nism of GAPDH regulation (i.e. CP12-independent)typical of GapB-containing isoforms, including nativeA2B2 and recombinant GapB (Baalmann et al., 1996; Liand Anderson, 1997; Sparla et al., 2002). In all plantspecies investigated, only the NADPH-dependentactivity of GAPDH is regulated (Steiger et al., 1971;Scagliarini et al., 1993). The regulatory mechanisminvolves thioredoxin and a number of metaboliceffectors, pyridine nucleotides and 1,3-bisphosphogly-cerate in particular, which affect the reversible aggre-gation of A2B2 tetramers into partially inactive A8B8oligomers (Pupillo and Giuliani Piccari, 1975; Trostet al., 1993; Baalmann et al., 1996). Similar to thiore-doxin regulation, the change of quaternary structureis also a CTE-dependent event (Baalmann et al., 1996;Li and Anderson, 1997; Sparla et al., 2002). The auton-omous regulation of AB-GAPDH in chloroplasts coex-ists with the regulatory supramolecular complexcontaining GAPDH, phosphoribulokinase, and CP12(Scheibe et al., 2002). The diverging features of thesetwo regulatory systems suggest distinct physiologicalroles connected to the light/dark status of the plant.

Here, we show that the redox sensitive, C-terminalextension of GapB is a versatile regulatory module,

Figure 3. Molecular mass estimates of eight GAPDH forms by sizeexclusion chromatography. Separations were performed on a Superdex200 column (HR 10/30) equilibrated with either 0.2 mM NADP (whitebars) or 0.2 mM NAD (black bars). Protein samples of 0.2 mL wereloaded for every run. For every sample, molecular mass estimationswere based on the elution volume of the absorbance peak at 280 nm.Mean values of duplicate experiments, with error bars representing SDs.

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which can induce novel regulatory functions whentransferred to GapA subunits. The chimeric mutantA(plusCTE) was found to be redox-sensitive and proneto aggregate in the presence of NAD, both typicalresponses of regulatory GAPDH isoforms (Sparla et al.,2002), but with certain peculiarities. Indeed, theNADH-activity of A(plusCTE) mutant was redox-sensitive rather than constitutive as in GapB. More-over, a comparison between oligomers of GapB,AB-GAPDH, and A(plusCTE) formed in the presenceof NAD (478, 759, and .1,800 kD, respectively; Fig. 3)suggests that A(plusCTE) produced aggregates ofmolecular size at least double that of AB-GAPDHand comprised of 30 subunits or more. Although theB(minCTE) sequence is 80% identical to GapA, clearlyB-subunits do not behave like A-subunits fused withCTE, and, judging from the relatively small size ofGapB aggregates (478 kD, B8), the CTE-dependentchange in quaternary structure is enhanced by thepresence of A-subunits.

GapA of spinach chloroplasts is the only type ofphotosynthetic GAPDH whose tridimensional struc-ture has been solved to date (Fermani et al., 2001;Falini et al., 2003; Sparla et al., 2004) and provides thebest approximation of the structure of GapB tetramers(CTE excluded). In GapA tetramers, A-subunits aresymmetrically distributed with respect to three per-pendicular axes (P,Q, and R; Fig. 4). Each subunit isconstituted by a coenzyme binding domain and a cat-alytic domain. Coenzyme molecules, either NADP orNAD, bind to the coenzyme site of each subunit andprotrude the catalytically active nicotinamide towardthe active site (Fig. 4).

The fine structure of recombinant spinach GapAcomplexed with NADP (Sparla et al., 2004) indicatesthat the side chains of Ser-188 and Arg-77 strictlyinteract with the 2#-phosphate of NADP. Similarinteractions are often involved in coenzyme recogni-tion in NADP-dependent dehydrogenases (Carugoand Argos, 1997), and in the case of photosyntheticGAPDH, they constitute the main basis for NADPrecognition. Thr-33 was also found to interact with the2#-phosphate in native A4-GAPDH (Fermani et al.,2001) but with a secondary role in coenzyme recogni-tion since its mutation only caused limited kineticeffects (Sparla et al., 2004). Arg-77 belongs to the samesubunit of the coenzyme site, whereas Ser-188 belongsto the opposite subunit in terms of R-axis and islocated in the middle of a long S-shaped loop (S-loop)that reciprocally links pairs of opposite subunits (Fig.4). In the presence of NAD instead of NADP, Arg-77and Ser-188 cannot interact with the coenzyme and theS-loop fails to connect the opposite subunits (Faliniet al., 2003). The functions of these and other aminoacids in recombinant GapB have been addressed bysite-directed mutagenesis.

In mutant B(R77A), the redox regulation is com-pletely abolished and NADPH activity is decreased tothe level of NADH activity (about 50 mmol min21

mg21). The role of Arg-77 thus seems to be crucial in

both coenzyme recognition and redox regulation.Mutant B(E362Q), too, is redox-insensitive, but theterminal residue Glu-362 is probably not involved incoenzyme discrimination since its NADPH-activity ishigher than the NADH-dependent one, as usual.Therefore, differences between mutant B(E362Q) andwild-type GapB become apparent only under oxidiz-ing conditions, which selectively depress the NADPH-activity of GapB without affecting the activity ofmutant B(E362Q). Mutants B(R77A) and B(E362Q)mimic two opposite redox states of regulatory GapB:B(R77A) is permanently inhibited and reminiscent ofoxidized GapB, B(E362Q) is fully ‘‘activated’’ (i.e.deinhibited) like reduced GapB. Mutant B(S188A) isintermediate in behavior, an intriguing parallel to theredox responses of A(plusCTE) construct (Fig. 2).

To explain all these results, we propose a modelin which the elevated NADPH-dependent GAPDHcatalysis depends on an optimal conformationadopted by the enzyme when NADPH is properlybound and held in place in the coenzyme site. In anyother case, catalytically less effective conformationswill be reached, as occurs during NAD(H)-dependentreactions and following CTE disulfide formation inthioredoxin-regulated GAPDH (with an approxi-

Figure 4. Ribbon model of the tridimensional structure of recombinantGapA from spinach (Sparla et al., 2004). Overview of the tetrameralong the molecular Paxis. The top-left subunit is colored dark gray; theother three are light gray. Four bound NADP molecules are schemat-ically represented. For the sake of clarity, in one subunit only (top-rightsubunit, colored light gray) NADP is colored black and the side chainsof Arg-77 (belonging to the same subunit) and Ser-188 (belonging to theR-axis related subunit, colored dark gray) are represented in sticks andballs. These two residues interact with the 2#-phosphate group ofNADP. Identical interactions occur in all other subunits (not shown).The image was produced using MOLSCRIPT (Kraulis, 1991).

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mately 2-fold drop in kcat of NADPH-dependent re-action; Table I).

That an optimal enzyme conformation is associatedwith maximal NADPH-activity has been known sincethe structure of A4-GAPDH complexed with NADPwas solved at 2.0 A resolution (Sparla et al., 2004). Asan example of a less efficient conformation, on theother hand, mutant S188A of GapA exhibits a de-creased NADPH-dependent activity and differs fromwild-type GapA in that the tetramer is expanded byabout 2 A in each direction (8% volume increase). Thefine structure of this mutant complexed with NADPrevealed that the R77-2#phosphate interaction waslacking, so the S-loops did not reciprocally link thepairs of R-axis related subunits resulting in a relaxedconformation of the tetramer (Sparla et al., 2004). Whyrelaxed-versus-compact conformations should corre-spond to low-versus-high NADPH activities is farfrom being clear, but structural and functional dataconcur to support our model of GAPDH redox regu-lation as represented in Figure 5. In this scheme, full

NADPH-activity of GAPDH is carried on by a compacttetramer (‘‘fast’’ conformation) in which the boundNADPH is primarily sensed via interactions betweenthe 2#-phosphate of the coenzyme and the side chainof Arg-77. GAPDH forms containing B-subunits withintact CTE have a fast conformation only under re-ducing conditions. Wild-type A4-GAPDH and mu-tants B(E362Q) (Fig. 5) and B(minCTE) (as well as CTE-cys mutants of GapB; Sparla et al., 2002) do so underany redox condition and are permanently activated. Inthe oxidized CTE, on the contrary, the formation ofa disulfide bridge forces the C-terminal Glu-362 toapproach Arg-77, thereby distracting it from interac-tions with the 2#-phosphate of NADP. Under theseconditions, Ser-188 and the opposite S-loop would quittheir interacting position with the 2#-phosphate andthe tetramer would relax to an expanded (‘‘slow’’)conformation that catalyzes a slower reaction. Thiskinetic behavior is found not only in all oxidized GapBforms (except B(E362Q) and CTE-cys mutants), butalso in mutants B(R77A) (this paper) and A(S188A)

Figure 5. Conceptual scheme of redox regulationof GapB subunits based on perturbed redoxregulation of site specific mutants. For the sakeof clarity, each case is represented by a singleGapB subunit. The change of location of thepositively charged residue R77 (which interactswith either the negative charges of NADP 2#-phosphate or of C-terminal residue E362) deter-mines a conformational change (slow or relaxed,fast or compact conformations). In this model,when R77 interacts with NADP (as in reducedGapB complexed with NADP or in B(E362Q)mutant under any redox condition) the confor-mation of the enzyme is compact (fast) and thecatalytic efficiency is maximal. On the contrary,when R77 does not interact with NADP (inNADP-binding GapB under oxidizing condi-tions, and in mutant B(R77A) under any redoxconditions), the enzyme conformation is relaxed(slow) and the catalytic efficiency is low. ResidueS188, which interacts with NADP in concert withR77, is not shown for the sake of clarity. In thepresence of NAD, there is no interaction betweenR77 and the coenzyme, and GapB displays lowactivity under any redox conditions, but theconformation of the tetramer might not be iden-tical to the slow conformation of inhibited GapBforms. Therefore, the slow conformation of NAD-binding GAPDH is differently represented.

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(Sparla et al., 2004) under any redox conditions.According to the model, the NADH-activity ofGAPDH is not regulated because Arg-77 and Ser-188do not interact with the coenzyme (Falini et al., 2003).However, in contrast to mutant A(S188A) complexedwith NADP (Sparla et al., 2004), the conformation ofNAD-complexed A4-GAPDH is not ‘‘expanded’’(Falini et al., 2003), although NADPH-dependentactivities of inhibited forms are similar to NADH-dependent ones. Other structural constraints mustexplain the low efficiency of the NADH-activity. Whiletridimensional structures of oxidized GapB forms arenot yet available, this work provides evidence that allGAPDH forms defective in coenzyme recognition doalso share a kinetically inefficient conformation spe-cifically affecting the NADPH-activity and possiblycorresponding to the expanded tetrameric conforma-tion of mutant A(S188A).

The complex regulation of photosynthetic GAPDHapparently reflects the important position occupied bythis enzyme in photosynthetic metabolism. Dark in-activation of GAPDH in vivo is needed to avoidfutile cycles with NADPH-producing metabolisms inchloroplasts such as the pentose phosphate pathway(Buchanan and Balmer, 2005). Accordingly, photosyn-thetic GAPDH activity might not be redox-regulatedin certain organisms such as diatoms (Liaud et al.,2000), which do not possess plastidic isoforms ofNADPH-producing enzymes of the pentose phos-phate pathway (Michels et al., 2005).

In land plants, dark inactivation of differentGAPDH isozymes (A4, A2B2) is achieved by differentmechanisms with a common structural basis. Regula-tion of A2B2-isozyme depends on the CTE of subunit-B, which works as a sensor of the thioredoxin redoxstate in the stroma. The NADPH activity of A2B2-GAPDH is inhibited by oxidized thioredoxins and byNAD, the latter promoting the formation of A8B8aggregates (Pupillo and Giuliani Piccari, 1975). Inphotosynthetic tissues, oxidized thioredoxins are moreabundant in darkness than in the light (Buchanan andBalmer, 2005). Under the same conditions, NAD in-creases at the expenses of NADP (Muto et al., 1980;Heineke et al., 1991), an effect that might be ascribed todark inactivation of NAD-kinase (Delumeau et al.,2000). The cooperation between oxidized thioredoxinsand NAD in inactivating GAPDH is demonstratedby the fact that mutants with no functional sensorfor thioredoxins [B(minCTE), this work; CTE-Cysmutants of GapB; Sparla et al., 2002] as well as nativeisozyme A4 (Scagliarini et al., 1998) are all insensitiveto NAD-induced inhibition/aggregation. Therefore,A4-GAPDH, which may account for up to 30% of thetotal GAPDH activity in chloroplasts of higher plants(Cerff, 1979), requires another mechanism for darkinactivation. This mechanism is more ancient in evo-lution than CTE-based GAPDH regulation and in-volves CP12 and PRK as partner proteins (Wedel andSoll, 1998). Formation of the GAPDH/CP12/PRKcomplex in chloroplasts results in inhibition of enzyme

activities and is favored in darkness (Scheibe et al.,2002). Oxidizing conditions and NAD promote com-plex formation in vitro (Graciet et al., 2004). The CTEof GapB derives in evolution from CP12 (Pohlmeyeret al., 1996) and both peptides share a similar C-ter-minal sequence, including the two redox-sensitive Cys(Sparla et al., 2002) and a C-terminal negativelycharged amino acid (Glu-362 in spinach GapB; Asp-74 in spinach CP12; Pohlmeyer et al., 1996). TheC-terminal portion of CP12 interacts with GAPDH(Wedel and Soll, 1998), and we propose that CP12 andCTE interact with GAPDH according to the same basicrules. Under oxidizing conditions, both CP12 and CTEhave a conformation dictated by the presence of aninternal disulfide bridge. This conformation forces aC-terminal negative charge (Glu-362 in CTE, Asp-74 inCP12) to approach Arg-77, thus distracting this resi-due from proper recognition of bound NADP. Theconsequence is enzyme inhibition, i.e. dark inactiva-tion in vivo of any GAPDH isozyme.

MATERIALS AND METHODS

Expression Vectors for Wild-Type andMutant GAPDH Subunits

The cDNA fragments coding for subunits GapA, GapB, deletion GapB

mutant lacking the C-terminal extension [B(minCTE); Baalmann et al., 1996],

and chimeric GapA mutant bearing the CTE at the C terminus [A(plusCTE)]

were transferred from pET-14 to pET-29 (Novagen) expression vectors. In

short, the inserts were excised from pET-14 with BamHI and NdeI, purified

from a 0.8% low melting agarose, and cloned into the BamHI/NdeI sites of the

expression vector pET-29. The correct DNA sequence of all inserts cloned in

pET-29 was controlled (Fig. 1).

Site-specific mutants B-E362Q, B-R77A, and B-S188A were constructed by

PCR. The following primers, with mutations indicated in bold, were used

according to the procedures of the QuickChange Site-Directed Mutagenesis

kit (Stratagene): E362Q-L, 5#-GAGGAGTGCAAACTTTACCAGTAAGGAT-

CCG-3#; E362Q-R, 5#-CGGATCCTTACTGGTAAAGTTTGCACTCCTC-3#;R77A-L, 5#-GGTTGTCTCTAACGCGGACCCTCTTAAACTTCCTTGGG-3#;R77A-R, 5#-CCCAAGGAAGTTTAAGAGGGTCCGCGTTAGAGACAACC-3#;S188A-L, 5#-CAGAGGCTGTGGGATGCTGCTCACAGGGACTTG-3#; S188A-R,

5#-CAAGTCCCTGTGAGCAGCATCCAACAGCCTCTG-3#.The 50-mL PCR reactions contained 125 ng of each primer and 50 ng of

template (pET29-GapB). Before transferring the plasmids into BL21(DE3)

cells, the coding sequence of each mutant was sequenced.

Heterologous Expression and Protein Purification

Escherichia coli cells, strain BL21(DE3), were transformed with the above

described constructs. Each E. coli BL21(DE3) strain harboring an expression

plasmid was cultured in 20 mL Luria-Bertani medium supplied with

kanamycin (50 mg/mL) at 37�C under vigorous shaking (130–150 rpm) for

16 to 18 h. Overnight cultures were transferred into 500 mL of fresh Luria-

Bertani medium in the presence of kanamycin and grown under shaking at

37�C until an optical density of 0.4 to 0.8 at 600 nm was reached. The

expression of recombinant proteins was induced by 0.4 mM isopropyl-b-D-

thiogalactopyranoside. Three hours after induction, cells were harvested by

centrifugation (10,000 g, 15 min). The pellet was washed with 25 mM

K-phosphate, pH 7.5, 1 mM EDTA, 10 mM b-mercaptoethanol, 1% (v/v)

glycerol (buffer A), and spun down again before storage at 270�C for no

longer than 2 d before use.

Cells were broken by lysozyme as described in Sparla et al. (2002). Shortly,

the pellet was resuspended in buffer A supplemented with 0.5 mM NADP and

10 mg mL21 lysozyme. After 15 min incubation at room temperature, 1 mg

mL21 DNase, 1 mg mL21 RNase, and 10 mM MgCl2 were added to the sample,

Regulation of Photosynthetic GAPDH

Plant Physiol. Vol. 138, 2005 2217

and incubation was prolonged for 20 min at 30�C. Cell debris was removed by

centrifugation (20000g, 20 min) and the clear supernatant recovered for

purification of recombinant proteins.

All recombinant proteins were purified by the same procedure. The

supernatant obtained after cell lysis was first loaded on a Q-Sepharose HP

16/10 anion-exchange column (Pharmacia) equilibrated with buffer A.

After thorough washing, bound proteins were eluted by 50 mL of linear

K-phosphate gradient. The composition of the gradient was 25 to 500 mM for

GapA and mutant B(minCTE), and 25 to 700 mM K-phosphate for any enzyme

form containing the CTE that provides additional negative charges [GapB,

A(plusCTE), B(S188A), B(R77A), and B(E362Q)]. Fractions containing

NADPH-dependent GAPDH activity were pooled and desalted in buffer

A. Samples were then loaded on a second anion-exchange column (MonoQ

HR 5/5; Pharmacia) equilibrated with buffer A. Proteins were eluted by 15 mL

of linear salt gradient (25–500 mM K-phosphate for GapA and B-CTE, and

25–700 mM K-phosphate for CTE-containing proteins). Fractions of 250 mL

were collected and assayed for GAPDH activity. Active fractions were pooled,

stored at 14�C, and used within the next 48 to 72 h.

Native AB-GAPDH was purified from spinach chloroplasts as previously

described (Sparla et al., 2002).

Protein concentration in purified protein samples was determined by the

Bradford method (Bradford, 1976) using bovine serum albumine as a standard.

Redox Titrations

For redox titration experiments, each enzyme form was desalted in 100 mM

Tricine-NaOH, pH 7.9 (Sparla et al., 2002). Enzyme samples (about 2 pmol

subunits) were incubated for 3 h at 25�C in the presence of an equimolar

amount of recombinant E. coli thioredoxin (Sigma, St. Louis) and 20 mM

dithiothreitol in different dithiol/disulfide ratios as described in Hutchinson

and Ort (1995). Standard NAD(P)H-GAPDH activity was assayed after

incubation in 50 mM Tris-HCl, 1 mM EDTA, 5 mM MgCl2, 3 mM 3-phospho-

glycerate, 2 mM ATP, 5 U mL21 phosphoglycerate kinase (from rabbit muscle),

and 0.2 mM NADPH. Oxidation of NADPH was followed at 340 nm with an

e340 of 6.23 mM21 (Falini et al., 2003). Activity data were fit by nonlinear

regression (CoStat, CoHort Software) to the Nernst equation with an n value

set at 2 (Hirasawa et al., 2000) and analyzed as described in Sparla et al. (2002).

Midpoint redox potentials (Em,7.9) are reported as mean values 6 SD of at least

three independent redox titrations performed under identical conditions and

individually analyzed.

Disulfide formation in redox-insensitive mutant B(E362Q) was checked by

labeling the enzyme with the fluorescent thiol-reagent monobromobimane

(Hutchinson and Ort., 1995) following the modified procedure of Hirasawa

et al. (1999).

Steady-State Kinetic Analysis

Steady-state kinetic analysis was accomplished by varying the concentra-

tion of NADPH between 250 and 5 mM (NADH between 300 and 10 mM) and

3-phosphoglycerate between 4 and 0.05 mM. The equilibrium concentration of

the 1,3-bisphosphoglycerate was calculated on the basis of phosphoglycerate

kinase Keq of 3.1 3 1024 (Bergmeyer, 1985). Each kinetic experiment typically

involved 70 single measurements of initial enzyme activity. Data were

analyzed by nonlinear regression (CoStat) using a Michaelis Menten equation:

v5 ðVmaxðappÞ 3 SÞ=ðKmðappÞ 1 SÞ;

where S is the concentration of varying substrate and Vmax(app) and Km(app) are

apparent kinetic constants. To obtain the limiting kinetic constants, the

Vmax(app) constants were interpolated by analogous nonlinear regression.

Kinetic parameters are given as mean 6 SD of at least three kinetic ex-

periments performed under identical conditions.

Size Exclusion Chromatography

The molecular mass of purified recombinant proteins was estimated by

size exclusion chromatography on a Superdex 200 HR 10/30 column as

described in Sparla et al. (2002). Briefly, samples were desalted in 100 mM

Tricine-NaOH, pH 7.9, and incubated on ice for 2 h in the presence of either

0.2 mM NADP (nonaggregating condition) or 0.2 mM NAD (aggregating

condition). The column was equilibrated in 50 mM Tris-HCl, pH 7.5, 1 mM

EDTA, 150 mM KCl, 14 mM b-mercaptoethanol, and 0.2 mM NADP or NAD.

Elution was performed in the same buffer and optical absorbance was mon-

itored at 280 nm. Fractions of 350 mL were collected and assayed for

NAD(P)H-GAPDH activity. Apparent molecular mass was calculated on the

basis of the elution volume of each 280-nm absorbance peak and the calibra-

tion of the column with ferritin (440 kD), catalase (232 kD), aldolase (158 kD),

ovalbumin (43 kD), chymotrypsinogen A (25 kD), and ribonuclease A

(13.7 kD) as standards (Pharmacia).

ACKNOWLEDGMENTS

We thank Simona Fermani, Giuseppe Falini, and Alberto Ripamonti for

helpful discussions.

Received March 2, 2005; revised April 22, 2005; accepted April 22, 2005;

published July 29, 2005.

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